Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

Bacterial strains and culture conditions

The wild-type O157 strain EDL933 (O157), a sequenced isolate implicated in human disease [21], was used in this study. We cultured O157 in Dulbecco Modified Eagle Medium-Low Glucose (DMEM; Gibco/lnvitrogen Corporation, Grand Island, NY), for the cell adherence assays described below. The rationale for the use of this culture medium was (i) to reflect the growth conditions used in the eukaryotic cell adherence assays; and (ii) to closely parallel the in vivo nutrient-limiting conditions, and conditions used to prepare the cattle-use approved, LEE protein based, anti-adhesion O157 vaccine. In addition, another wild-type O157 strain 86–24 (86–24), its isogenic mutant (86-24eae Δ10) negative for Intimin, and this mutant complemented with the plasmid pEB310 (86-24eae Δ10(pEB310)) expressing Intimin, were also tested in the adherence assay [22]. The 86–24 strain and its derivatives were obtained from Dr. A. D. O’Brien, Uniformed Services University of the Health Sciences, Bethesda, MD.

We also cultured O157 in DMEM for proteomic analysis. Specifically, an overnight culture of the wild-type O157 strain in Luria-Bertani (LB) broth was pelleted and washed with sterile phosphate buffered saline (PBS; pH 7.4), and subcultured to an initial OD₆₀₀ of 0.05 in fresh DMEM. After incubation at 37 °C with shaking at 250 rpm to an OD₆₀₀ of 0.8 to 1.0, cells were harvested by centrifugation at 7,000 rpm, 15 min at 4 °C. Cells were washed three times with an equal volume of sterile PBS (pH 7.4), and processed to obtain cell lysate and pellet fractions for proteomic analysis as previously described [23].

O157-RSE cell adherence inhibition assay: (i) in the presence of pooled anti-LEE proteins, anti-intimin and anti-H7 antisera

Adherence of O157 to the RSE cells was previously demonstrated and developed into an adherence assay in our laboratory [5]. In this study, the ability of pooled, rabbit polyclonal antisera to interfere with and inhibit O157 adherence to RSE cells was evaluated. Specifically, antisera generated against the recombinant LEE-encoded proteins, Tir, EspA and EspB, and Intimin, in rabbits (National Animal Disease Center Stocks), was pooled. Rabbit antisera targeting the O157 flagellar antigen H7 (Difco Laboratories, Inc., Detroit, MI) was also mixed into the pooled antisera, which was then tested at 1:5 and 1:10 dilutions. Specificity was confirmed by reacting each antiserum against both O157 cell lysates and the cognate protein in western blotting experiments (data not shown). Rabbit sera (Sigma-Aldrich, St. Louis, MO) from healthy animals (normal rabbit sera), at a 1:5 dilution, was used as a control.

(ii) In the presence of anti-Intimin antisera alone

To specifically evaluate the role of intimin, the rabbit anti-Intimin antisera was evaluated separately for its ability to prevent O157 adherence to RSE cells at 1:5 and 1:10 dilutions.

Each of the RSE adherence assays was conducted in 8 technical and 2 biological replicates as described previously [5], with minor modifications, as follows. RSE cells were washed and resuspended in 1 ml Dulbecco Modified Eagle Medium – No Glucose (DMEM-NG) ± 2.5% D + Mannose, in 16 x 100 mm glass tubes, to a final concentration of 10⁵ cells/ml. Although Type 1 fimbriae are not expressed by O157, we included D + Mannose in parallel assays to cover any hitherto unknown transient expression especially in mutant strains. Bacterial pellets from overnight cultures in DMEM, incubated at 37°C without aeration, were resuspended in sterile saline with or without antisera (‘no sera’ control), and incubated at 37°C for 30 min. The bacteria-antibody mix was then added to the RSE cells suspension to final bacteria:cell ratio of 10:1, and the mixture incubated with aeration (37°C, 110 rpm, for 4 h). At the end of 4 h, the mixture was pelleted and washed thoroughly, once with 14 ml DMEM-NG, and twice with 14 mls of sterile, distilled water (dH₂O) before reconstituting in 100 μl dH₂O. Eight 2 μl drops of this suspension were placed on Polysine (Thermo Scientific Pierce) slides and dried overnight under direct light to quench non-specific fluorescence, before fixing in cold 95% ethanol for 10 min. The slides were then stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target O157 and the RSE cell cytokeratins as described previously [5]. Each experiment was then done in duplicate.

O157 adherence patterns on RSE cells were recorded as diffuse, or aggregative (clumps) for all positive interactions that involved direct association with the cells [5]. Scattered bacteria and bacterial micro-colonies not adhering to cell membranes were considered to be negative for adherence to the epithelial cells [5]. A total of 100–160 well dispersed RSE cells (10–20 cells per drop or chamber) were analyzed per slide as described previously [5]. The percent RSE cells with and without bacteria adhering to them were determined [5]. If more than 50% of RSE cells had >10 bacteria attached, the adherence was recorded as strongly positive. For >50% RSE cells with 1–10 adherent bacteria, the adherence was recorded as moderately positive. For less than 50% RSE cells with 1–5 adherent bacteria, the result was recorded as non-adherent.

O157-HEp-2 cell adherence inhibition assay

The role played by LEE-encoded proteins and Intimin in the adherence of O157 to HEp-2 cells, has already been defined previously [22] and hence, this assay was used for comparative reasons. The assay was conducted as described previously [5] except that, the washed bacterial pellets were incubated with or without antisera (‘no sera’ control), at 37°C for 30 min, prior to addition to the HEp-2 cells. Both the pooled antisera and anti-intimin antisera, as described above, were used at dilutions ranging from 1:5 to 1:100 in these assays. Each assay was conducted in duplicate, and in 3–6 chambers of the chamber slides per run. Slides were stained with 1% toluidine blue, or with fluorescence-tagged antibodies that specifically target O157 and the HEp-2 cell actin filaments as described previously [5] and adherence patterns recorded as for RSE cells (see above).

Adherence of 86–24, 86-24eae Δ10, and 86-24eae Δ10(pEB310), to RSE and HEp-2 cells

Wild-type 86–24 and its mutant derivatives were used to verify the role of Intimin directly and compare the results with that of the O157 adherence inhibition assays. This assay was conducted, recorded, as previously described and done in the absence of any antisera [5].

OneDimensional (1D) SDS-PAGE liquid chromatography tandem mass spectrometry (GeLC-MS/MS)

Top down proteomic analysis was done at the Harvard Partners Center for Genetics and Genomics, Cambridge, Massachusetts. O157 cell pellet and lysate fractions were concentrated using spin filters (MW cutoff 5000 Daltons; Vivascience Inc., Englewood, NY), fractionated on 1D SDS-PAGE, and digested in-gel with trypsin prior to tandem mass spectrometry (MS/MS) as described previously [23]. The rationale for incorporating a 1D SDS-PAGE fractionation step is that this modification reduces complexity of protein mixtures, permits a larger dynamic range of protein identification, and allows for significantly better reproducibility [24, 25].

For mass spectrometry (MS), samples were subjected to three different runs on an LCQ DECA XP plus Proteome X workstation (LCQ) from Thermo Finnigan as described earlier [23, 26]. For each run, 10 μL of each reconstituted sample was injected with a Famos Autosampler, and the separation was done on a 75 μm (inner diameter) x 20 cm column packed with C₁₈ media running at a flow rate of 0.25 μl/min provided from a Surveyor MS pump with a flow splitter with a gradient of water, 0.1% formic acid and then 5% acetonitrile, 0.1% formic acid (5%-72%) over the course of 480 min (8.0 hour run). Between each set of samples, a standard of a 5 Angiotensin mix of peptides (Michrom BioResources) was run to ascertain column performance, and to observe any potential carryover that might have occurred. The LCQ was run in a top five configuration, with one MS scan and five MS/MS scans. Dynamic exclusion was set to 1 with a limit of 30 seconds.

Peptide identifications were made using SEQUEST (Thermo Finnigan) through the Bioworks Browser 3.2, as described previously [23]. Sequential database searches were performed using the O157 strains EDL933 and Sakai FASTA database from European Bioinformatics Institute http://www.ebi.ac.uk/newt/display using static carbamidomethyl-modified cysteines and differential oxidized methionines. These protein databases (Escherichia coli (strain Sakai/O157:H7/RIMD 0509952/EHEC) – Tax ID: 386585 and Escherichia coli (strain EDL933/ATCC 700927/O157:H7/EHEC) – Tax ID: 155864) have a total of 10,737entries. A reverse O157 strain EDL933 FASTA database was spiked in to provide noise and determine validity of the peptide hits, so that known and theoretical protein hits can be determined without compromising the statistical relevance of all the data [26]. The MS data was searched with a 2-Dalton window on the MS precursor with a 0.8 Dalton on the fragment ions. Peptide score cutoff values were chosen at cross-correlation values (Xcorr) of 1.8 for singly charged ions, 2.5 for doubly charged ions, and 3.0 for triply charged ions, along with delta rank scoring preliminary cutoff (deltaCN) values of 0.1, and cross-correlation normalized values (RSp) of 1. The cross-correlation values chosen for each peptide assured a high confidence match for the different charge states, while the deltaCN values ensured the uniqueness of the peptide hit. The RSp value of 1 ensured that the peptide matched the top hit in the preliminary scoring. At these peptide filter values, very few reverse database hits were observed, which permitted a higher confidence in the few single peptide protein identifications. Furthermore, single hit proteins were manually validated to ensure relevance.

Bioinformatics

Cellular location of proteins was determined using amino acid sequences of cognate proteins in the O157 sequence databases at http://www.ncbi.nlm.nih.gov/protein. In addition, extracytoplasmic proteins were verified for the presence of signal sequences using the program SignalP 3.0 at http://www.cbs.dtu.dk/services/SignalP, and subcellular localization of other proteins confirmed using the PSORT/PSORT-B program (http://psort.nibb.ac.jp/). Putative functions were determined by querying the Conserved Domain Database (CDD) at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi Protein components of the O157 DMEM-proteome with adhesion potential were shortlisted using Vaxign, a reverse vaccinology based vaccine target prediction and analysis system at http://www.violinet.org that utilizes the SPAAN algorithm [27].